微小RNA214调节破骨细胞分化的分子机制及在骨质疏松治疗中的作用研究

Osteoporosis is a major medical problem that threatens the health of the elderly, but its molecular mechanism is still unclear. Our previous study has suggested that miR-214 levels were significantly elevated in bone specimens of osteoporosis patients. Bone resorption was much higher in osteoclast-specific miR-214 transgenic mice compared to wild type group. Osteoclast differentiation was suppressed by miR-214 inhibitor in vitro. These data suggest that miR-214 play an important role in osteoclast differentiation and osteoporosis. Therefore, we intend to construct an osteoclast-specific miR-214 knockout mice to investigate the regulating function of miR-214 in osteoporosis. Cell biology and molecular biology loss-function and gain-function methods will be used to explore the molecular mechanism of miR-214 in regulating osteoclast differentiation in vitro and in vivo. Finally, an osteoclast specific nucleic acid delivery system will be used to silence endogenous miR-214 in osteoporotic mice for observing the therapeutic effects of miR-214 inhibitor. This study will provide a theoretical support and experimental basis for osteoporosis nucleic acid therapy.

骨质疏松是威胁老年人健康的重大医学问题，其发病分子机制尚不清楚。课题组前期研究发现骨质疏松患者骨标本中微小RNA214（miR-214）水平显著升高。预实验发现破骨细胞特异性miR-214转基因小鼠骨吸收能力增强，而miR-214抑制剂则削弱了体外破骨细胞的分化进程。上述证据暗示miR-214可能是促进破骨细胞分化的调节因子，并在骨质疏松发生过程中有重要作用。因此我们拟构建miR-214破骨细胞特异性敲除小鼠，以确认miR-214与骨质疏松的关系。综合运用loss-fuction和gain-function细胞生物学和分子生物学手段，在小鼠体内和体外研究miR-214对破骨细胞分化的调节作用，并深入探索其分子机制，进而利用靶向破骨细胞的核酸递送系统静默骨质疏松小鼠的内源性miR-214，观察miR-214抑制剂的治疗效果。本研究将为骨质疏松核酸药物治疗提供理论支持和实验基础。

骨质疏松是威胁老年人健康的重大医学问题，但是其发病的分子机制尚不清楚。miRNA是一种在转录后水平调控蛋白表达的小分子非编码RNA，广泛参与疾病的发生发展过程。课题组前期研究发现骨质疏松患者骨标本中miR-214水平显著升高，miR-214 可以促进体外破骨细胞的分化进程。通过本项目的实施，我们发现miR-214过表达可显著促进破骨细胞分化，而miR-214抑制剂可导致破骨细胞分化受阻。进而，我们构建了破骨细胞特异性的miR-214敲除小鼠，组织学检测发现该小鼠骨量升高。microCT检测显示该小鼠骨骼微结构异常，骨小梁数量增加、厚度均增加，分离度降低。课题组通过生物信息学分析，发现Pten是miR-214的潜在靶基因。Western blot检测显示，miR-214过表达Pten蛋白水平下降，而使用抑制剂降低内源性miR-214后Pten的蛋白水平增加。双荧光酶报告实验表明miR-214通过与Pten 3’UTR直接作用实现对Pten的负向转录后调控。进而，课题组分析了人骨质疏松样品中miR-214表达水平与骨吸收的相关性，结果表明miR-214与骨质疏松患者的骨吸收正相关，而与骨质疏松患者的骨量负相关。进而，课题组在小鼠OVX骨质疏松模型中，破骨细胞靶向性递送miR-214抑制剂，结果发现骨吸收活性下降，骨丢失得到了显著抑制。综上所述，我们明确了miR-214对破骨细胞分化的调节作用和分子机制，为后续的转化应用提供了实验支持。在本项目的资助下，课题组于ACS Nano、Nature Communications、Theranostics等国际知名刊物共发表SCI论文9篇，其中影响因子大于5的SCI论文7篇，影响因子大于10的SCI论文2篇。已培养研究生3名。项目负责人获国家自然科学基金优秀青年基金资助。